A computational approach to decipher chromatosome structure determinants

In eukaryotic cells, DNA transcription, replication and repair events are controlled by the regulation of DNA compaction mechanisms that determine the open and closed chromatin states. Nucleosomes are the basic DNA packaging units of chromatin. The nucleosome core (NC) consists of a core histone protein octamer with approximately two tight superhelical turns of DNA wrapped around it. The NC is extended at its entry and exit points by linker DNA (L-DNA) and a linker histone (LH) protein binds between the two L-DNA arms to form a chromatosome. The dyad is the single DNA base pair between the nucleosome entry and exit points determining the symmetry axis and is used to define the position of LH binding to a nucleosome. For LH - nucleosome binding, previous studies indicate both on- and off-dyad binding modes, as well as different LH orientations. Thus, the molecular determinants of the structure of LH – nucleosome complex and the dynamics of LH – nucleosome binding are not fully understood. The aim of the research described here was to obtain an atomic-detail level understanding of chromatosome formation. Analysis of the experimentally determined structures of LH – nucleosome complexes showed that instead of a single 3D structure, an ensemble of structures of LH – nucleosome complexes exists. To understand the distribution of these ensembles, normal mode analysis (NMA), standard and accelerated molecular dynamics (MD & AMD) and Brownian dynamics (BD) simulations were applied to LH, nucleosome and chromatosome systems. MD and AMD simulations showed that the globular domain of the LH (LH GD) prefers to be in its closed form in solution. Upon nucleosome binding, the LH GD structure transformed to an open structure due to hydrophobic interactions with the L-DNA of the nucleosome. Additionally, LH GD binding constrained the flexibility of the L-DNA and affected the directions of movement of the L-DNA arms. BD simulations indicated that various chromatosome configurations were possible depending on LH GD sequence and L-DNA opening angles. These findings suggest that LH – nucleosome binding is mediated by a combination of conformational selection and induced fit mechanisms. Further BD simulations show that chromatosome configurations were affected by single point mutations in the LH GD and varied for different LH isoforms. My results indicate that by making specific single point mutation exchanges, it is possible to swap LH – nucleosome configurations among different LH GD isoforms. Similar shifts were observed in chromatosome configuration upon introduction of post translational modifications (PTMs) in the LH GD. I applied BD simulations to compute dissociation rate constant (koff) values and compare them with previously reported fluorescence recovery after photobleaching (FRAP) data on the binding of various LH mutants to chromatin. The results of the BD simulations correspond with the relative trends in measured FRAP recovery half-times (t50) of LH – chromatin binding of various LH mutants. The results thus enable the interpretation of the FRAP data in terms of a physical model of LH – nucleosome binding. My thesis provides detailed insights into the structure, dynamics and kinetics of chromatosome formation in eukaryotes. The results presented in this work can guide further experiments on the sequence determinants of LH – nucleosome binding

Single-Molecule Investigation of Chromatin-Associated Factors in Genome Organization and Epigenetic Maintenance

The central dogma of biology has laid the foundation for understanding gene expression through the mechanisms of transcription and translation. However, another layer of eukaryotic gene regulation lies in the complex structure of chromatin. This scaffold of structural proteins and enzymatic regulators determines what genes are expressed at what times, leading to cell differentiation, cell fate, and often disease. Currently, the field of chromatin biology has relied on basic biochemistry and cellular assays to identify key epigenetic regulators and their role in genomic maintenance. For this thesis work, I have developed a biophysical platform to study chromatin-associated factors at the single-molecule level (Chapter 2). This methodology allows us to extract key mechanistic details often obscured by standard bulk methodologies. Using this platform, we posed the question of how epigenetic factor, Polycomb repressive complex 2 (PRC2) engages with chromatin (Chapter 3). PRC2 is a major epigenetic machinery that maintains transcriptionally silent heterochromatin in the nucleus and plays critical roles in embryonic development and oncogenesis. It is generally thought that PRC2 propagates repressive histone marks by modifying neighboring nucleosomes in a strictly linear progression. However, the behavior of PRC2 on native-like chromatin substrates remains incompletely characterized, making the precise mechanism of PRC2-mediated heterochromatin maintenance elusive. Our understanding of this process was limited by the resolution of structural techniques that fail to identify PRC2-binding modes on long chromatin substrates. In short, we found direct evidence that PRC2 can simultaneously engage nonadjacent nucleosome pairs. The demonstration of PRC2\u27s ability to bridge noncontiguous chromosomal segments furthers our understanding of how Polycomb complexes spread epigenetic modifications and compact chromatin. In addition to this single-molecule chromatin binding technology, I also created a singlemolecule platform harnessing correlative force and fluorescence microscopy to assay the material properties of phase separated condensates (Chapter 2). This assay combined methodology to visualize condensate formation at the single-molecule level, in addition to optical trapping of individual droplets to investigate their material properties. Utilizing this technology, we interrogated the role of linker histone H1 (Chapter 4). The linker histones are the most abundant group of chromatin-binding proteins that bind and organize eukaryotic chromatin. However, roles for the diverse and largely unstructured H1 proteins beyond chromatin compaction remain unclear. We used correlative single-molecule force and fluorescence microscopy to directly visualize the behavior of H1 on DNA under different tensions. Unexpectedly, our results show that H1 preferentially coalesces around nascent, relaxed singlestranded DNA. In vitro bulk assays confirmed that H1 has a higher propensity to form phaseseparated condensates with single-stranded DNA than with double-stranded DNA. Furthermore, we dissected the material properties of different H1:DNA condensates by controlled droplet fusion with optical tweezers, and found that increased DNA length and GC content result in more viscous, gel-like H1 condensates. Overall, our findings suggest a potential role for linker histones to sense and coacervate single-stranded nucleic acids in the nucleus, forming reaction hubs for genome maintenance. This work also provides a new perspective to understand how various H1 subtypes and disease-associated mutations affect chromatin structure and function. In summary, we have gained a greater understanding of the biophysical basis for chromatin regulation by both PRC2 and histone H1. Both of the biophysical platforms created for these studies can be applied to various new targets in chromatin biology. They will enable the investigation of a multiplicity of binding interactions, regulatory mechanisms, and material properties of protein-nucleic acid complexes (Chapters 5 & 6). I believe single-molecule techniques will become a major toolset to study chromatin biology, identifying the intricacies and interactions between epigenetic factors and our genome

Functional characterization of the Saccharomyces cerevisiae chromatin remodeler INO80

Knowing the explicit locations of nucleosomes in a genome is a pre-requisite for understanding the regulation of genes. Predominantly at regulatory active promoter sites, regular spaced arrays phased at reference points shape the chromatin landscape. In eukaryotic cells ATP-dependent chromatin remodeler align nucleosomes at reference points and are pivotal in the formation of the stereotyped promoter pattern. Chromatin remodeler of the ISWI, CHD, SWI/SNF and INO80 family convert energy derived from ATP hydrolysis to operate on their nucleosomal substrates to accomplish nucleosome spacing, eviction and editing reactions. Recent structural elucidations provided mechanistic insights into how chromatin remodelers engage their nucleosomal substrates (Eustermann et al., 2018, Aramayo et al., 2018, Willhoft et al., 2018, Ayala et al., 2018, Farnung et al., 2017, Wagner et al., 2020, Yan et al., 2019, He et al., 2020, Han et al., 2020) and brought about a unifying DNA wave mechanism underpinning ATP-dependent DNA translocation by chromatin remodeling complexes (Yan and Chen, 2020). Understanding how phased arrays of equally spaced nucleosomes are generated by chromatin remodelers represents an ultimate long-term goal in chromatin biology. What remains unclear is the underlying mechanism that directs nucleosome positioning by chromatin remodelers in absolute terms. How do ATP-dependent chromatin remodelers generate phased arrays of regularly spaced nucleosomes? How are the distances between nucleosomes and phasing sites and between adjacent nucleosomes set? Is DNA shape read-out part of nucleosome positioning driven by chromatin remodelers? Do remodelers have intrinsic ruler-like elements that set spacing and phasing distances? The aim of this thesis was to delineate whether, and if so, what type of genomic information is read by a remodeler in the stereotypic placement of nucleosomes at physiological sites, and how the remodeler activities fit into the unifying framework of ATP-dependent DNA translocation mechanism of chromatin remodelers. To gain an insight into nucleosome positioning driven by Saccharomyces cerevisiae (S.c.) ATP-dependent chromatin remodelers, a combination of a minimalistic genome-wide in vitro reconstitution system, biochemical analysis, high-resolution structures and structure-guided mutagenesis of the S.c. INO80 model system was applied. Findings of this work would have an impact on the mechanistic understanding of nucleosome positioning driven by ATP dependent chromatin remodelers based on the ruler concept that has been described earlier for the ISW1a chromatin remodeler (Yamada et al., 2011). The ISW1a, Chd1 and ISW2 remodelers demonstrated “clamping” activity and used ruler elements to set 1 Abstract distances with a defined linker length (21-26 bp at all densities, 12-13bp at all densities, 54-58 bp at low/medium densities, respectively). Mutagenesis of the INO80 model system identified and tuned the INO80 ruler element, which is comprised of the Ino80_HSA domain of the ARP module, the NHP10 module and Ino80 N-terminal residues. Regularly spaced symmetrical arrays were generated at the Reb1 reference point sites as well as at BamHI-introduced dsDNA break sites. Nucleosome positioning on the genomic sequences of S. c., Schizosaccharomyces pombe (S.p.) as well as Escherichia coli (E.coli) showed no significant differences. Mutagenesis of residues located within the Ino80_HSA domain established a causal link between nucleosome positioning by INO80 and DNA shape read-out by the INO80_HSA domain. The spacing and phasing distances generated by ATP-dependent chromatin remodelers point towards a remodeler-intrinsic ruler activity that is independent of underlying DNA sequences and can be sensitive to nucleosome density. This study measured linker lengths set by remodeler-intrinsic ruler-like functionalities in absolute terms, which will be instrumental to dissect contributions from individual remodelers in nucleosome positioning in vivo. This provides the starting point to understand how remodeler-driven nucleosome dynamics direct stable steady-state nucleosome positions relative to DNA bound factors, DNA ends and DNA sequence elements. Sequence-dependent DNA shape features have been mainly associated with binding of transcription factors as well as general regulatory factors and more static DNA binding events. This study augments the general description of nucleosome positioning sequences for chromatin remodelers by establishing nucleosome positioning motifs based on DNA shape analysis. This study provides an intriguing framework to implement DNA shape read-out in the tracking mechanism of DNA-translocating machineries

Regulation of DNA replication by chromatin structure in mammalian cells

Tesis Doctoral inédita leída en la Universidad Autónoma de Madrid, Facultad de Ciencias, Departamento de Biología Molecular. Fecha de lectura: 21-09-2017Esta tesis tiene embargado el acceso al texto completo hasta el 21-03-201

Functional characterization of the Saccharomyces cerevisiae chromatin remodeler INO80

Knowing the explicit locations of nucleosomes in a genome is a pre-requisite for understanding the regulation of genes. Predominantly at regulatory active promoter sites, regular spaced arrays phased at reference points shape the chromatin landscape. In eukaryotic cells ATP-dependent chromatin remodeler align nucleosomes at reference points and are pivotal in the formation of the stereotyped promoter pattern. Chromatin remodeler of the ISWI, CHD, SWI/SNF and INO80 family convert energy derived from ATP hydrolysis to operate on their nucleosomal substrates to accomplish nucleosome spacing, eviction and editing reactions. Recent structural elucidations provided mechanistic insights into how chromatin remodelers engage their nucleosomal substrates (Eustermann et al., 2018, Aramayo et al., 2018, Willhoft et al., 2018, Ayala et al., 2018, Farnung et al., 2017, Wagner et al., 2020, Yan et al., 2019, He et al., 2020, Han et al., 2020) and brought about a unifying DNA wave mechanism underpinning ATP-dependent DNA translocation by chromatin remodeling complexes (Yan and Chen, 2020). Understanding how phased arrays of equally spaced nucleosomes are generated by chromatin remodelers represents an ultimate long-term goal in chromatin biology. What remains unclear is the underlying mechanism that directs nucleosome positioning by chromatin remodelers in absolute terms. How do ATP-dependent chromatin remodelers generate phased arrays of regularly spaced nucleosomes? How are the distances between nucleosomes and phasing sites and between adjacent nucleosomes set? Is DNA shape read-out part of nucleosome positioning driven by chromatin remodelers? Do remodelers have intrinsic ruler-like elements that set spacing and phasing distances? The aim of this thesis was to delineate whether, and if so, what type of genomic information is read by a remodeler in the stereotypic placement of nucleosomes at physiological sites, and how the remodeler activities fit into the unifying framework of ATP-dependent DNA translocation mechanism of chromatin remodelers. To gain an insight into nucleosome positioning driven by Saccharomyces cerevisiae (S.c.) ATP-dependent chromatin remodelers, a combination of a minimalistic genome-wide in vitro reconstitution system, biochemical analysis, high-resolution structures and structure-guided mutagenesis of the S.c. INO80 model system was applied. Findings of this work would have an impact on the mechanistic understanding of nucleosome positioning driven by ATP dependent chromatin remodelers based on the ruler concept that has been described earlier for the ISW1a chromatin remodeler (Yamada et al., 2011). The ISW1a, Chd1 and ISW2 remodelers demonstrated “clamping” activity and used ruler elements to set 1 Abstract distances with a defined linker length (21-26 bp at all densities, 12-13bp at all densities, 54-58 bp at low/medium densities, respectively). Mutagenesis of the INO80 model system identified and tuned the INO80 ruler element, which is comprised of the Ino80_HSA domain of the ARP module, the NHP10 module and Ino80 N-terminal residues. Regularly spaced symmetrical arrays were generated at the Reb1 reference point sites as well as at BamHI-introduced dsDNA break sites. Nucleosome positioning on the genomic sequences of S. c., Schizosaccharomyces pombe (S.p.) as well as Escherichia coli (E.coli) showed no significant differences. Mutagenesis of residues located within the Ino80_HSA domain established a causal link between nucleosome positioning by INO80 and DNA shape read-out by the INO80_HSA domain. The spacing and phasing distances generated by ATP-dependent chromatin remodelers point towards a remodeler-intrinsic ruler activity that is independent of underlying DNA sequences and can be sensitive to nucleosome density. This study measured linker lengths set by remodeler-intrinsic ruler-like functionalities in absolute terms, which will be instrumental to dissect contributions from individual remodelers in nucleosome positioning in vivo. This provides the starting point to understand how remodeler-driven nucleosome dynamics direct stable steady-state nucleosome positions relative to DNA bound factors, DNA ends and DNA sequence elements. Sequence-dependent DNA shape features have been mainly associated with binding of transcription factors as well as general regulatory factors and more static DNA binding events. This study augments the general description of nucleosome positioning sequences for chromatin remodelers by establishing nucleosome positioning motifs based on DNA shape analysis. This study provides an intriguing framework to implement DNA shape read-out in the tracking mechanism of DNA-translocating machineries

Chromatin and Epigenetics

Genomics has gathered broad public attention since Lamarck put forward his top-down hypothesis of 'motivated change' in 1809 in his famous book "Philosophie Zoologique" and even more so since Darwin published his famous bottom-up theory of natural selection in "The Origin of Species" in 1859. The public awareness culminated in the much anticipated race to decipher the sequence of the human genome in 2002. Over all those years, it has become apparent that genomic DNA is compacted into chromatin with a dedicated 3D higher-order organization and dynamics, and that on each structural level epigenetic modifications exist. The book "Chromatin and Epigenetics" addresses current issues in the fields of epigenetics and chromatin ranging from more theoretical overviews in the first four chapters to much more detailed methodologies and insights into diagnostics and treatments in the following chapters. The chapters illustrate in their depth and breadth that genetic information is stored on all structural and dynamical levels within the nucleus with corresponding modifications of functional relevance. Thus, only an integrative systems approach allows to understand, treat, and manipulate the holistic interplay of genotype and phenotype creating functional genomes. The book chapters therefore contribute to this general perspective, not only opening opportunities for a true universal view on genetic information but also being key for a general understanding of genomes, their function, as well as life and evolution in general

Meiosis

Meiosis is the key process underlying sexual reproduction in eukaryotes, occurring in single-celled eukaryotes and in most multicellular eukaryotes including animals and most plants. Thus meiosis is of considerable interest, both at the scientific level and at the level of natural human curiosity about sexual reproduction. Improved understanding of important aspects of meiosis has emerged in recent years and major questions are starting to be answered, such as: How does meiosis occur at the molecular level, How did meiosis and sex arise during evolution, What is the major adaptive function of meiosis and sex. In addition, changing perspectives on meiosis and sex have led to the question: How should meiosis be taught. This book proposes answers to these questions, with extensive supporting references to the current literature

Towards the development of a novel proteomic tool to map trimethyllysine marks on histones via a Hofmann-type chemical modification.

One of the key epigenetic processes for transcriptional control is dynamic post-translational modification (PTM) of histone proteins. Dysregulation of these epigenetic mechanisms have been linked to the aetiology of human diseases such as cancer and neurological disorders. To decipher the epigenetic pathways leading to the development of disease and to gain insight into the function of post-translational modifications of histones, a great interest was taken on the genome-wide mapping of epigenetic marks. The most commonly used method to profile PTMs on histones is the ChIP-seq technique (chromatin immunoprecipitation followed by DNA sequencing), which is based on the use of antibodies to target and enrich specific epigenetic marks. However, the dependence of ChIP-seq on antibodies constitutes a significant limitation due to possible cross-reactivity and poor specificity of those antibodies towards the targeted epigenetic marks. In addition, the ChIP-seq technique only allows the mapping of single epigenetic marks to genomic loci. Here we present a novel antibody-free chemical biological tool ‘CLICK-seq’ to map combinatorial histone lysine trimethylation marks on intact mono-nucleosomes. It is based on the selective modification of trimethyllysine residues on histone proteins to obtain an alkene functionality on these substrates, which enables the introduction of affinity tags via a thiol-ene click chemistry for selective enrichment. This work demonstrates diverse chemical approaches taken towards the elimination of trimethylamine in quaternary ammonium substrates to achieve the formation of an alkene under mild and ‘protein-compatible’ conditions. With the development of a novel reaction - Pd-catalysed Hofmann-type elimination – the removal of trimethylamine was achieved at low temperature on model small molecules (albeit in a low yield). In the second project, a novel design for acid-cleavable cross-linkers was developed to simultaneously achieve the cross-linking of histone protein complexes and the subsequent chemical labelling of histone lysine residues upon acid-catalysed cleavage. This work portrays our efforts towards the synthesis of a small library of acid-cleavable cross-linkers.Open Acces

Studying and manipulating chromatin motion in mammalian cells

Histone modifications such as methylation and acetylation are known to be key determinants in the regulation of gene expression, but little is known about how higher order chromatin structures, and their spatial organisation in the nucleus, can control gene expression. This remains a key question in addressing the role of spatial organisation in genomic function.If changes in nuclear position have a role in gene expression, chromatin within the cell must be able to move distances that would accommodate this. In the first part of my PhD I investigated the range of chromatin motion in living human cells. E.coli Lac operator arrays inserted into the human genome and visualised using Lac repressor protein fused to GFP, are able to move up to 2-3 pm over the period of two hours, distances greater than previously reported and similar to motion observed in yeast. I have also determined whether the position of a locus is conserved from one cell cycle to the next by following cells through mitosis. From this analysis it was concluded that although some aspects of positioning were conserved, loci position was established anew each cell cycle.Although I have shown that chromatin mobility is quite constrained within the nucleus, proteins associated with chromatin have been shown to be highly mobile. I have investigated the effect of different factors that might affect the mobility of linker histones using fluorescence recovery after photobleaching. I have shown that while Su(Var)3-9, responsible for tri-methylation of Lysine 9 on histone 3, and MeCP2, a DNA methylation binding protein, have no effect on linker histone mobility, the methylation of DNA does. In the absence of DNA methylation, linker histones are more tightly bound to the chromatin fibre.In humans it is well established that chromosomes have a gene-density related radial organisation within the cell nucleus. I have mapped the radial position of mouse chromosomes in ES cells to determine if a similar pattern of organisation exists. My results suggest there may be a loose correlation between chromosome size and position within the mouse genome, but not gene density. Furthermore differentiation iii of mouse ES cells, induced changes in the position of some chromosomes, suggesting that gene expression may have a role in chromosome position.Although correlations in nuclear position and expression have been seen in many model organisms, only in budding yeast has there been direct experimental confirmation that position can control gene expression. To determine directly if nuclear position can regulate gene expression in the mouse I aimed to artificially tether a gene to the edge of the mouse nucleus. Arrays of Lac operator sequences were inserted into or near genes. To tether genes to the nuclear periphery, Lac repressor was fused to the integral membrane proteins emerin or LAP2ß. I have shown that these fusion proteins can transiently anchor transfected Lac-operator containing plasmids to the nuclear periphery of mouse cells and that this silences gene expression from these plasmids. Anchoring of endogenous mouse genes was also investigated